j pharmacol exp ther 2013 sukhtankar 11 22

1521-0103/346/1/11–22$25.00 http://dx.doi.org/10.1124/jpet.113.203984THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 346:11–22, July 2013Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

Effects of Spinally Administered Bifunctional Nociceptin/Orphanin FQ Peptide Receptor/m-Opioid Receptor Ligandsin Mouse Models of Neuropathic and Inflammatory Pain

Devki D. Sukhtankar, Nurulain T. Zaveri, Stephen M. Husbands, and Mei-Chuan KoDepartment of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan (D.D.S.); Astraea Therapeutics, LLC,Mountain View, California (N.T.Z.); Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (S.M.H.);and Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina(M.-C.K.)

Received February 12, 2013; accepted May 6, 2013

ABSTRACTNociceptin/orphanin FQ peptide receptor (NOP) agonists produceantinociceptive effects in animal models after spinal administrationand potentiate m-opioid receptor (MOP)-mediated antinociception.This study determined the antinociceptive effects of spinally adminis-tered bifunctional NOP/MOP ligands and the antinociceptive func-tions of spinal NOP and MOP receptors in mice. Antinociceptiveeffects of bifunctional NOP/MOP ligands BU08028 [(2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol]and SR16435 [1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one] were pharmacologically comparedwith the putative bifunctional ligand buprenorphine, selective NOPagonist SCH221510 [3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol] andselectiveMOPagonistmorphine in neuropathic and inflammatory pain models. Addi-tionally, the degree of tolerance development to the antiallodyniceffects of SR16435 and buprenorphine were determined after

repeated intrathecal administration. Our data indicated thatBU08028 and SR16435 were more potent than morphine andSCH221510 in attenuating nerve injury-induced tactile allodyniaand inflammation-induced thermal hyperalgesia. Coadministra-tion of receptor-selective antagonists further revealed that bothNOP and MOP in the spinal cord mediated the antiallodyniceffects ofBU08028andSR16435, but intrathecal buprenorphine-induced antiallodynic effects were primarily mediated by MOP.Repeated intrathecal administration of SR16435 resulted inreduced and slower development of tolerance to its antiallodyniceffects compared with buprenorphine. In conclusion, both NOPand MOP receptors in the spinal cord independently drive anti-nociception in mice. Spinally administered bifunctional NOP/MOP ligands not only can effectively attenuate neuropathic andinflammatory pain, but also have higher antinociceptive potencywith reduced tolerance development to analgesia. Such ligandstherefore display a promising profile as spinal analgesics.

IntroductionNociceptin/orphanin FQ receptor (NOP), the fourth mem-

ber of the opioid family with similarities in localization andcellular actions to the classic opioid receptors, is implicated inthe modulation of pain responses (Lambert, 2008; Largent-Milnes and Vanderah, 2010; Calo and Guerrini, 2013). TheNOP system in the brain and spinal cord is upregulated inrodents under neuropathic and inflammatory pain conditions(Jia et al., 1998; Briscini et al., 2002). In rodents, activation ofspinal NOP is shown to produce antinociception in acute,

neuropathic, and inflammatory pain (Tian et al., 1997; Haoet al., 1998; Obara et al., 2005). In nonhuman primates,however, NOP agonists produce antinociception by systemicas well as spinal administration andwithoutm-opioid-associatedside effects (Ko et al., 2009; Hu et al., 2010). Overall, thesestudies indicate that one of the main sites of antinociceptiveactions for NOP agonists is the spinal cord. This makes NOPa potential target for spinal analgesia.Spinal NOP and m-opioid receptor (MOP) independently

drive antinociception in preclinical pain models. In rodents,spinal administration of NOP agonists potentiated morphine-induced antinociception in the absence of motor dysfunction(Tian et al., 1997; Courteix et al., 2004; Reiss et al., 2008). Innonhuman primates as well, NOP activation potentiatedMOP-mediated antinociception, devoid of side effects such asrespiratory depression (Hu et al., 2010; Cremeans et al.,2012). These studies emphasize the importance of bifunctional

This study was supported by the National Institutes of Health NationalInstitute of Arthritis and Musculoskeletal and Skin Diseases [Grant R01-AR-059193]; and by the National Institutes of Health National Institute on DrugAbuse [Grant R01-DA-032568]. The content is solely the responsibility of theauthors and does not necessarily represent the official views of the NationalInstitutes of Health.

dx.doi.org/10.1124/jpet.113.203984.

ABBREVIATIONS: BU08028, (2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol; J-113397, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; MOP,m-opioid receptors; %MPE, percentage of the maximum possible effect; NOP, nociceptin/orphanin FQ receptors; Ro 64-6198, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one; SCH221510, 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol; SR16435, 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one.

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ligands, which can simultaneously activate both NOP andMOP receptors. Such ligands may have a wider therapeuticwindow and the ability to treat severe pain conditions. Ad-ditionally, coactivation of NOP and MOP may also result inslower tolerance development because both receptor pools areused to a lesser degree to achieve analgesia. Collectively,bifunctional NOP/MOP ligands may have a promising clinicalvalue and need to be investigated in animal models.Bifunctional ligands with varying degrees of affinity and

efficacy at NOP and MOP were tested in rodents for theirantinociceptive effects after systemic administration (Spagnoloet al., 2008; Toll et al., 2009; Khroyan et al., 2011b). However,it is not known how NOP and MOP receptors in the spinalcord contribute to antinociception when bifunctional ligandsare directly injected in the spinal cord. One of the putativebifunctional ligands is buprenorphine, which has high bindingaffinity and agonist activity at MOP (Lewis, 1985). Althoughbuprenorphine has extremely low binding affinity for NOP(Toll et al., 1998), systemic buprenorphine-induced antinoci-ception was enhanced in rodents when NOP antagonists weresystemically administered (Lutfy et al., 2003; Khroyan et al.,2009), indicating the suppression of buprenorphine’s anti-nociception by the NOP component. However, it is unclearhow spinal NOP directly modulates antinociceptive effects ofbuprenorphine. It is therefore important to pharmacologicallycharacterize antinociceptive actions of bifunctional NOP/MOPligands and buprenorphine after acute and repeated spinaladministration.Our study determined the function of NOP versus MOP

receptors in the spinal cord to produce antinociception againstneuropathic pain, relative to inflammatory pain, by usingselective and bifunctional ligands. In particular, we studiedbifunctional ligands with high affinity and partial agonistactivity at NOP andMOP—BU08028 [2-(N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl)-3,3-dimethylpentan-2-ol] and SR16435 [1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one]—but without agonist activity at d- and k-opioid receptors(Khroyan et al., 2007, 2011a), and we compared themwith theselective NOP andMOP agonists as well as buprenorphine. Inaddition, we compared potential tolerance development to theantiallodynic effects of SR16435 and buprenorphine, whichhave similar duration of actions, after repeated intrathecaladministration.

Materials and MethodsAnimals

Male ICR mice weighing 25–30 g were used (Harlan Industries,Indianapolis, IN). Mice were housed five per cage with food and waterad libitum and a 12-hour light/dark cycle under standard laboratoryconditions. All procedures were conducted in accordance with theUniversity Committee on the Use and Care of Animals at theUniversity of Michigan (Ann Arbor, MI) and the Guide for Care andUse of Laboratory Animals as adopted and promulgated by theNational Institutes of Health (Bethesda, MD).

Chronic Constriction Injury

Nerve injury was induced by ligation of the sciatic nerve using themethod developed by Bennett and Xie (1988) modified for mice.Briefly, mice were anesthetized with ketamine (80 mg/kg i.p.)/xylazine (12 mg/kg i.p.). An incision was made between the gluteus

superficialis and biceps femoris muscles in the right leg to expose thesciatic nerve. Four chromic gut ligatures were tied loosely around thesciatic nerve at 1-mm intervals in such a way that the epineural bloodflow was occluded but not arrested. The wound was closed by suturingthe muscles and the skin.

Carrageenan-Induced Paw Inflammation

Mice were lightly anesthetized with 3% isoflurane and receivedan intraplantar injection of 50 ml l-carrageenan (2%; Sigma-Aldrich,St. Louis, MO) dissolved in saline in the right hind paw.

Drug Administration

Morphine sulfate, naltrexone, buprenorphine (National Instituteon Drug Abuse, Bethesda, MD), and SR16435 (Astraea Therapeutics,Mountain View, CA) were dissolved in sterile water. SCH221510 [3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol] (Tocris Bioscience, Bristol, UK), BU08028 (University of Bath, Bath,UK), and J-113397 [1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (Tocris Biosci-ence) were dissolved in 1:1:8 ratio of dimethylsulfoxide (DMSO), Tween80, and sterile water.

All drugs were administered intrathecally in the volume of 5 ml aspreviously described elsewhere (Fairbanks, 2003). Briefly, the mousewas secured by a firm grip on the pelvic girdle, and the drugs wereinjected by lumbar puncture between the L5/L6 vertebrae using a 30-gauge needle attached to a 10-ml Hamilton syringe.Mice in the controlgroup received an intrathecal injection of the appropriate vehicle.

Behavioral Analyses

Tactile Allodynia. Tactile allodynia was measured in mice withchronic constriction injury as paw withdrawal thresholds in responseto probing with calibrated von Frey filaments in a manner describedby Chaplan et al. (1994). Mice were habituated for 45 minutes insuspended cages designed with wire-mesh floors. von Frey filamentswith buckling weights ranging from 0.04 to 2 g were then appliedperpendicularly to the plantar surface of the ipsilateral paw andheld for 5 seconds. A positive response was indicated by a sharpwithdrawal of the paw. For eachmouse, the testing began with a vonFrey filament corresponding to the weight of 0.4 g. If the animalmade a positive response, the next filament with lower force wasapplied. If the response was negative, the next filament with higherforce was used. Each response was recorded, and the experimentended once the animal had made five responses after the firstpositive response.

The 50% paw withdrawal threshold was determined by the Dixonnonparametric method (Dixon, 1980). If the animal made fourconsecutive positive responses, a score of 0.04 g was assigned. If theanimal made four consecutive negative responses, a score of 2 g wasassigned. Baseline values for paw withdrawal thresholds wereobtained in all mice before the induction of nerve injury. Changesin tactile allodynia in response to drug treatment were determined2 weeks after the nerve injury was induced.

Thermal Hyperalgesia

Thermal hyperalgesia was measured in mice with paw inflamma-tion induced by the intraplantar carrageenan injection. Thermalhyperalgesia was measured using the hot plate method, with thesurface temperature maintained at 52°C. Mice were placed on the hotplate, and the latency for withdrawal of the carrageenan-treated pawfrom the hot plate wasmeasured. A cutoff latency of 30 seconds (s) wasused to avoid tissue damage. Baseline values for paw withdrawallatencies were obtained in all mice before the induction of pawinflammation. Changes in thermal hyperalgesia in response to drug

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treatment were determined 2 hours after the injection of carrageenanin the paw.

Experimental Design

Mice with nerve injury developed significant tactile allodyniawithin 2 weeks after the chronic constriction injury of the sciatic nerveand showed reduced withdrawal thresholds of the ipsilateral pawin response to von Frey filaments. No changes were observed inwithdrawal thresholds of the contralateral paw. Hence, all tests wereconducted by probing only the ipsilateral paw. Mice that received theintraplantar injection of carrageenan developed thermal hyperalgesia2 hours later and showed significant reduction in the paw withdrawallatency of the ipsilateral paw in response to noxious heat. Once thepredrug values for paw withdrawal thresholds and paw withdrawallatencies were obtained, drugs were intrathecally administered.Separate groups of animals (n 5 6–8 per group) were used to studyeach dosing condition. All behavioral experiments were conducted byexperimenters blinded to the condition.

The first part of the study was conducted to characterize the potency,duration, and effectiveness of selective NOP agonist SCH221510,selective MOP agonist morphine, and the bifunctional ligandsBU08028, SR16435, and buprenorphine against neuropathic andinflammatory pain. Mice with nerve injury-induced tactile allodyniareceived an intrathecal injection of SCH221510 (0.3–10 mg), morphine(0.3–10 mg), BU08028 (0.03–1 mg), SR16435 (0.1–3 mg), buprenorphine(0.1–3 mg), or vehicle. Similarly, the mice with carrageenan-inducedthermal hyperalgesia received an intrathecal injection of SCH221510(0.1–3.0 mg), morphine (0.1–3.0 mg), BU08028 (0.001–0.1 mg), SR16435(0.03–1 mg), buprenorphine (0.01–1 mg), or vehicle.

The doses of morphine and SCH221510 were selected based on theprevious studies in rodent pain models using NOP or MOP agonists(Hao et al., 1998; Sounvoravong et al., 2004; Obara et al., 2005). Wehypothesized that for a compound with the ability to activate NOPandMOP receptors it may be ideal to use doses that were 3 to 10 timeslower than those of SCH221510 and morphine. Once the doses thatproduced approximately 50% antinociception were obtained, the dose-response curves for all compounds were characterized. Because nodifference was noted in the nociceptive thresholds of the mice that hadreceived intrathecal saline or the mixture of dimethylsulfoxide(DMSO), Tween 80, and water, the data were pooled for all vehiclegroups. After the mice had received the drugs or vehicle, theirbehavioral analyses were conducted at time points of 0.5, 1, 2, 3, 4, 24,and 48 hours.

The second part of the study was conducted to determine thefunction of spinal NOP and MOP receptors to produce antiallodyniaagainst neuropathic pain. Doses of SCH221510 (10 mg), morphine(10 mg), BU08028 (1 mg), SR16435 (3 mg), and buprenorphine (3 mg),which produced near maximal antiallodynia, were selected. Antago-nists, which included naltrexone (0.3–3.0 mg) and J-113397 (0.3–3.0mg), or a single solution containing 3 mg of both naltrexone andJ-113397, were administered intrathecally as a 10-minute pretreat-ment. Mice that received intrathecal morphine were administerednaltrexone (0.3–3.0 mg) or J-113397 (3 mg). Mice that receivedintrathecal SCH221510 were administered J-113397 (0.3–3.0 mg)or naltrexone (3 mg). Highest doses of antagonists naltrexone (3 mg),J-113397 (3 mg), or the combination of both antagonists (3 mg),which completely blocked the antiallodynic effects of the selectiveagonists, were chosen as a 10-minute pretreatment to BU08028,SR16435, or buprenorphine. A group of mice in each agonist treat-ment also received the intrathecal pretreatment of the respectivevehicle.

On the day of testing, the predrug values for paw withdrawalthresholds were measured. The mice then were injected with theassigned antagonist doses. Selective or bifunctional ligands wereadministered 10 minutes later. Paw withdrawal thresholds wereagain measured at 30 minutes after the agonist treatment todetermine the effects of antagonist pretreatment. A separate group

of mice received all antagonist treatments alone followed by in-trathecal vehicle administration. No changes in nociceptive thresh-olds were observed. Hence, the data are not shown.

The third part of the study was conducted to determine the rate anddegree of tolerance development to the antiallodynic effects ofbuprenorphine (3 mg) and SR16435 (3 mg) in mice with neuropathicpain. Doses that produced near maximal antiallodynia with sim-ilar duration of action were chosen for the tolerance study. Forbuprenorphine and SR16435, the area under the curve was calculatedfor the percentage of the maximum possible effect (%MPE) of theirantiallodynic effects up to 48 hours at 3 mg. Based on the area underthe curve, the schedule of drug administration was determined.SR16435 was injected three times a day separated by 4 hours,whereas buprenorphine was injected 2 times a day separated by 6hours. Both compounds were administered for 5 days, and the pawwithdrawal thresholds were measured each day at 0.5 hours aftertheir administration.

This schedule of repeated drug administration may be morerelevant to a clinical setting in which the drug is readministeredonce its analgesic effects start to diminish. Previous studies have useda dosing schedule of more than two times a day for intrathecaladministration (Davis and Inturrisi, 1999; Choi et al., 2004; Hopkinset al., 2004). No signs of distress or overt pain behaviors wereobserved in these mice after the repeated intrathecal injections.

Statistical Analysis

Antiallodynic and antihyperalgesic effects were quantified in eachanimal as %MPE at each time point and drug dose. The followingformula was used to quantify %MPE: % MPE 5 [(Postdrug value fora behavioral response (g or s) 2 Predrug value for a behavioralresponse/(Cutoff value 2 Predrug value for a behavioral response) �100. If the animal did not respond to any von Frey filament or did notrespond before 30 seconds, a score of 100% MPE was assigned.

The mean values (6 S.E.M.) were calculated from individualanimals for all behavioral end points. Multiple comparisons weremade using repeatedmeasures two-way analysis of variance. Post hocanalyses were conducted using the Bonferroni test. Comparisons ofdata at a single time point were made using one-way analysis ofvariance followed by the Dunnett test. P , 0.05 was consideredstatistically significant for all tests. The 50% effective dose (ED50) plus95% confidence interval (CI) values were determined from the %MPEof each drug at the 0.5-hour time point.

ResultsFigure 1 illustrates the time course and antiallodynic

effects of intrathecal SCH221510 (0.3–10 mg) and morphine(0.3–10 mg) on tactile allodynia in mice with chronic con-striction injury. Before the surgical manipulation, all micemaintained high paw withdrawal thresholds close to thecutoff value (1.86 0.1 g). Statistically significant reduction inthe paw withdrawal thresholds was measured at 2 weeksafter the nerve injury was induced (0.096 0.01 g). IntrathecalSCH221510 produced an increase in the paw withdrawalthresholds, thereby increasing %MPE for antiallodynia indose-dependent [(F(4,25)5 4.4, P, 0.05] and time-dependent[(F(6,150) 5 43.2, P , 0.05] manners. Similarly, intrathecalmorphine produced antiallodynic effects in dose-dependent[F(4,25) 5 79.2, P , 0.05] and time-dependent [F(6,150) 552.2, P , 0.05] manners. Both SCH221510 and morphine at10 mg showed near maximal %MPE of 91 6 2 and 89 6 6,respectively, elevating the pawwithdrawal thresholds close totheir presurgery values. For both drugs, the peak antiallo-dynic effect was observed at 0.5 hours after drug administra-tion. These effects of SCH221510 lasted for 3 hours; for

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morphine, they lasted for 2 hours. Nociceptive thresholds didnot change in the mice that had received the intrathecalinjection of a vehicle.Figure 2 illustrates the time course and antihyperalgesic

effects of intrathecal SCH221510 (0.1–3 mg) and morphine(0.1–3 mg) on thermal hyperalgesia in mice with carrageenan-induced paw inflammation. Before the induction of inflamma-tion, all mice showed high paw withdrawal latencies (21.8 60.1 seconds). Significant reduction in the paw withdrawallatencies was measured at 2 hours after the carrageenaninjection (9.26 0.1 seconds). Intrathecal SCH221510 producedan increase in the paw withdrawal latencies and %MPE for itsantihyperalgesic effects in dose-dependent [F(4,25) 5 5.9, P ,0.05] and time-dependent [(F(6,150)5 11.6,P, 0.05]manners.Similarly, intrathecal morphine produced antihyperalgesiceffects in dose-dependent [F(4,25) 5 14, P , 0.05] and time-dependent [F(6,150) 5 42.2, P , 0.05] manners. BothSCH221510 and morphine at 3 mg showed near maximal%MPE of 89 6 5 and 95 6 5, respectively, elevating the pawwithdrawal latencies close to their precarrageenan values.For both drugs, the peak antihyperalgesic effect was observedat 0.5 hours after drug administration. These effects of

SCH221510 lasted for 3 hours whereas for morphine theylasted for 1 hour. Paw withdrawal latencies did not changein the mice that had received the intrathecal injection ofa vehicle.Figure 3 illustrates the time course and antiallodynic

effects of intrathecal BU08028 (0.03–1 mg), SR16435 (0.1–3mg), and buprenorphine (0.1–3 mg) on tactile allodynia in micewith chronic constriction injury. BU08028 caused an in-crease in the paw withdrawal thresholds and %MPE for itsantiallodynic effects in dose-dependent [F(4,35) 5 13.5, P ,0.05] and time-dependent [F(6,210) 5 26, P , 0.05] manners.The peak effect (%MPE of 98 6 1) of BU08028 at 1 mg wasobserved within 0.5 hours and lasted up to 24 hours. SR16435also dose-dependently [F(4,25) 5 32.9, P , 0.05] and time-dependently [F(6,150) 5 69.2, P , 0.05] increased the pawwithdrawal thresholds and%MPE for its antiallodynic effects.The peak effect (%MPE of 92 6 6) of SR16435 at 3 mg wasobserved within 0.5 hours and lasted up to 3 hours. Similarly,buprenorphine increased paw withdrawal thresholds and%MPE for its antiallodynic effects in dose-dependent [F(4,25)523.9, P, 0.05] and time-dependent [F(6,150)5 21.2, P, 0.05]manners. The peak effect (%MPE of 886 4) of buprenorphine

Fig. 1. Antiallodynic effects of intrathecal administration of SCH221510 and morphine in mice with neuropathic pain. Effects of SCH221510 (A) andmorphine (C) on paw withdrawal thresholds (g). Percentage of maximum possible effect of SCH221510 (B) and morphine (D) for attenuating tactileallodynia. BL, baseline values before induction of nerve injury. Pre-, predrug values before intrathecal administration of drugs. Behavioral responseswere measured at 0.5, 1, 2, 3, 4, 24, and 48 hours after drug administration. Each value represents the mean 6 S.E.M. (n = 6). Symbols representdifferent dosing conditions in different groups of mice. *Statistically significant difference from the vehicle controls (s, 0 mg) (P , 0.05).


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at 3 mg was observed within 0.5 hours and lasted up to 4hours.Figure 4 illustrates the time course and antihyperalgesic

effects of intrathecal BU08028 (0.001–0.1 mg), SR16435(0.03–1.0 mg), and buprenorphine (0.01–1.0 mg) on thermalhyperalgesia in mice with carrageenan-induced paw inflam-mation. BU08028 caused an increase in the paw withdrawallatencies and %MPE for its antihyperalgesic effects in dose-dependent [F(5,30) 5 46.8, P , 0.05] and time-dependent[F(6,180) 5 22.6, P , 0.05] manners. The peak effect (%MPEof 876 5) of BU08028 at 0.1 mg was observed within 0.5 hoursand lasted up to 24 hours. SR16435 also produced dose-dependent [F(4,25) 5 16.7, P , 0.05] and time-dependent[F(6,150) 5 17.5, P , 0.05] increase in the paw withdrawallatencies and %MPE for its antihyperalgesic effects. Peakeffect (%MPE of 89 6 6) of SR16435 at 1 mg was observedwithin 0.5 hours and lasted up to 4 hours. Buprenorphinecaused an increase in the paw withdrawal latencies and%MPE for its antihyperalgesic effects in dose-dependent[F(6,29) 5 35.7, P , 0.05] and time-dependent [F(6,174) 5 30,P, 0.05] manners. The peak effect with %MPE of 986 2 and

956 5 was observed within 0.5 hours after the administrationof 0.3 and 1 mg of buprenorphine, respectively. These effectslasted up to 4 hours.Figure 5 compares the antihypersensitive (antiallodynic

and antihyperalgesic) potencies of intrathecally administeredNOP- and MOP-related ligands in neuropathic and inflam-matory pain. For both neuropathic and inflammatory painmodalities, SCH221510 and morphine showed similar poten-cies. BU08028, SR16435, and buprenorphine were morepotent than SCH221510 and morphine in blocking neurop-athic and inflammatory pain. In particular, all drugs weremore potent for their antihyperalgesic effects against in-flammatory pain as compared with their antiallodynic effectsagainst neuropathic pain.Figure 6 illustrates the effects of intrathecally administered

NOP and MOP antagonists as a pretreatment on antiallo-dynic activity of SCH221510 (10 mg) and morphine (10 mg) at0.5 hours in mice with neuropathic pain. NOP antagonistJ-113397 (0.3–3.0 mg) attenuated the antiallodynic effectsof SCH221510 in a dose-dependent [F(3,20) 5 54.5, P ,0.05] manner. Complete inhibition of SCH221510-induced

Fig. 2. Antihyperalgesic effects of intrathecal administration of SCH221510 and morphine in mice with inflammatory pain. Effects of SCH221510 (A)andmorphine (C) on pawwithdrawal latencies (sec). Percentage of maximum possible effect of SCH221510 (B) andmorphine (D) for attenuating thermalhyperalgesia. BL, baseline values before induction of paw inflammation. Pre-, predrug values before intrathecal administration of drugs. Behavioralresponses were measured at 0.5, 1, 2, 3, 4, 24, and 48 hours after drug administration. Each value represents the mean 6 S.E.M. (n = 6). Symbolsrepresent different dosing conditions in different groups of mice. *Statistically significant difference from the vehicle controls (s, 0 mg) (P , 0.05).


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antiallodynia was observed at 3 mg of J-113397. MOPantagonist naltrexone at 3 mg did not significantly blockthe antiallodynic effects of SCH221510. Pretreatment with

naltrexone (0.3–3.0 mg) dose-dependently [F(3,20) 5 42.9,P , 0.05] attenuated the antiallodynic activity of morphine.Complete inhibition of morphine-induced antiallodynia was

Fig. 3. Antiallodynic effects of intrathecal administration of BU08028, SR16435, and buprenorphine in mice with neuropathic pain. Effects of BU08028 (A),SR16435 (C), and buprenorphine (E) on paw withdrawal thresholds (g). Percentage of maximum possible effect of BU08028 (B), SR16435 (D), andbuprenorphine (F) for attenuating tactile allodynia. BL, baseline values before induction of nerve injury. Pre-, predrug values before intrathecal administrationof drugs. Behavioral responses were measured at 0.5, 1, 2, 3, 4, 24, and 48 hours after drug administration. Each value represents the mean 6 S.E.M. (n = 8).Symbols represent different dosing conditions in different groups of mice. *Statistically significant difference from the vehicle controls (s, 0 mg) (P , 0.05).

Fig. 4. Antihyperalgesic effects of intrathecal administration of BU08028, SR16435, and buprenorphine in mice with inflammatory pain. Effects ofBU08028 (A), SR16435 (C), and buprenorphine (E) on paw withdrawal latencies (sec). The %maximum possible effect of BU08028 (B), SR16435 (D), andbuprenorphine (F) for attenuating thermal hyperalgesia. BL, baseline values before induction of paw inflammation. Pre-, predrug values beforeintrathecal administration of drugs. Behavioral responses were measured at 0.5, 1, 2, 3, 4, 24, and 48 hours after drug administration. Each valuerepresents the mean6 S.E.M. (n = 8). Symbols represent different dosing conditions in different groups of mice. *Statistically significant difference fromthe vehicle controls (s, 0 mg) (P , 0.05).


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observed at 3 mg of naltrexone. J-113397 at 3 mg did notsignificantly block the antiallodynic effects of morphine.Figure 7 illustrates the effects of intrathecally administered

naltrexone (3 mg), J-113397 (3 mg) or their coadministration(3 mg) on the antiallodynic activity of BU08028 (1 mg), SR16435(3 mg) and buprenorphine (3 mg) at 0.5 hours in mice withneuropathic pain. There were statistically significant differ-ences among the dosing conditions for BU08028 [F(3,23) 532.6, P , 0.05], SR16435 [F(3,23) 5 68.2, P , 0.05], andbuprenorphine [(3,23) 5 48.3, P , 0.05]. Pretreatment withnaltrexone or J-113397 when administered individually par-tially but significantly blocked antiallodynic effects of BU08028and SR16435. When naltrexone and J-113397 were coadminis-tered, complete blockade of antiallodynic effects of BU08028and SR16435 was observed. Naltrexone alone or coadminis-tration of naltrexone and J-113397 significantly attenuatedbuprenorphine’s antiallodynic effects. However, J-113397when administered individually failed to block antiallodyniceffects of buprenorphine.Figure 8 compares the effects of repeated intrathecal admin-

istration of SR16435 (3 mg) and buprenorphine (3 mg) on thedevelopment of tolerance to their antiallodynic effects in mice

with neuropathic pain at 0.5 hours after their administration.There were statistically significant differences among the dosingconditions for SR16435 [F(4,25) 5 3.2, P , 0.05] andbuprenorphine [F(4,25)5 6.5, P, 0.05]. A small change of 12%in the %MPE of SR16435was observed on day 5 compared withday 1. For buprenorphine, an approximately 35% changein %MPE was observed on day 5 compared with day 1.

DiscussionThe first part of the study demonstrated the antihypersen-

sitive effects of spinally administered bifunctional NOP/MOPligands in comparison with the selective NOP and MOPagonists in neuropathic and inflammatory pain modalities inmice (Figs. 1–5). Antihypersensitive effects of intrathecallyadministered NOP agonist SCH221510 and MOP agonistmorphine were similar in terms of their potency, durationof action, and efficacy. Both SCH221510 and morphineeffectively blocked nerve injury–induced tactile allodynia andinflammation-induced thermal hyperalgesia in dose-dependentmanners. Neither SCH221510 nor morphine affected the motorfunction in mice at antihypersensitive doses, as similarly

Fig. 5. Comparison of the antinociceptive potencies of intrathecally administered NOP- and MOP-related ligands in neuropathic and inflammatorypain at 0.5 hours after drug administration. Dose-response curves for of BU08028, SR16435, buprenorphine, morphine, and SCH221510 for theirantiallodynic effects against neuropathic pain (A) and antihyperalgesic effects against inflammatory pain (B). Different symbols represent differentdrugs. (C) Comparison of the antinociceptive effects as ED50 + 95% confidence interval values of BU08028, SR16435, buprenorphine, morphine, andSCH221510: s represents each drug’s ED50 against neuropathic pain, and u represents each drug’s ED50 against inflammatory pain.


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reported in neuropathic rats after intrathecally administeredNOP agonist Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one](Obara et al., 2005). Despite differences in the ligand-bindingspecificities, NOP and MOP drive antinociception throughsimilar mechanisms. Activation of NOP receptors blockscAMP production and calcium currents and activatespotassium currents, which ultimately inhibit the nocicep-tive neurotransmission (Meunier et al., 1995; Reinscheidet al., 1995).Previous studies have reported that intrathecal adminis-

tration of both NOP and MOP agonists can produce anti-nociceptive and antihypersensitive effects, indicating thatNOP and MOP receptors in the spinal cord independentlydrive these effects. The present study is the first to demonstrateantihypersensitive effects of intrathecally administered bi-functional NOP/MOP ligands in neuropathic and inflammatory

pain. Intrathecal BU08028 and SR16435, both of which bindto NOP and MOP with high affinity (Ki 5 2–8 nM) and partialefficacy as measured by guanosine 59-O-(3-[35S]thio)triphos-phate functional assay (20–50% stimulation) (Khroyan et al.,2007, 2011a), were more potent than intrathecal SCH221510or morphine for their antihypersensitive effects (Fig. 5). Forinstance, BU08028 was 10 to 20 times more potent thanmorphine in producing antiallodynic effects in neuropathicpain as per the ED50 values (0.09 versus 1.6 mg) or the doserequired to produce full antiallodynia (1 versus 10 mg). Thedifference in potency was also observed against carrageenan-induced thermal hyperalgesia. BU08028 and SR16435 effec-tively produced antihyperalgesic effects at doses 3 to 10 timeslower than those of morphine or SCH221510. Overall, thesefindings indicate that not only with improved potency, bi-functional ligands with partial agonist action are also aseffective as selective full agonists.

Fig. 6. Effects of NOP and MOP antagonists on antiallodynic effects of intrathecal SCH221510 (10 mg) and morphine (10 mg) in neuropathic pain.Antagonists were administered intrathecally 10 minutes before SCH221510 or morphine. Changes in SCH221510-induced increase in the pawwithdrawal thresholds (A) and the percentage of maximum possible antiallodynic effect (B) after J-113397, naltrexone, or vehicle pretreatment at 0.5hours after intrathecal SCH221510. Right panels: changes in morphine-induced increase in the paw withdrawal thresholds (B) and the percentage ofmaximum possible antiallodynic effect (D) after naltrexone, J-113397, or vehicle pretreatment at 0.5 hours after intrathecal morphine. *Statisticallysignificant difference from the vehicle control (P , 0.05).


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The second part of the study determined the role of spinalNOP and MOP in modulating neuropathic pain (Figs. 6 and 7).Pretreatment with intrathecal administration of MOP antag-onist naltrexone dose-dependently attenuated antiallodyniceffects of morphine. Similarly, pretreatment with intrathecaladministration of NOP antagonist J-113397 dose-dependentlyattenuated antiallodynic effects of SCH221510. However,the dose of naltrexone that completely abolished morphine’santiallodynic effects did not attenuate the antiallodyniceffects of SCH221510, indicating selective MOP inhibitionof naltrexone. Similarly, the largest dose of J-113397 didnot block antiallodynic activity of morphine, indicating theselective NOP inhibition of J-113397. When naltrexone (3 mg)and J-113397 (3 mg) were administered individually, anapproximately 50% reduction in the antiallodynic effects ofBU08028 or SR16435 was observed. More importantly, co-administration of naltrexone and J-113397 completely blockedthe antiallodynic effects of BU08028 and SR16435 (Fig. 7).These data indicate that both NOP andMOP receptors in thespinal cord were activated to achieve antiallodynia inducedby intrathecal administration of bifunctional NOP/MOP li-gands. These findings are consistent with their in vitro profile,which indicates that BU08028 and SR16435 activate bothNOP andMOP receptors whenmeasured by the guanosine 59-O-(3-[35S]thio)triphosphate functional assay (Khroyan et al.,2007, 2011a). However, previous studies in rodents havesuggested that the antinociceptive effects of systemicallyadministered BU08028 and SR16435 in the tail flick assaywere mainly mediated by MOP receptors and that thepretreatment with systemic administration of NOP antag-onists enhanced the ascending part of the dose-response

curve for the antinociceptive effects of BU08028 andSR16435 (Khroyan et al., 2009, 2011a).Such discrepancies in receptor mechanisms underlying

antinociception after spinal versus systemic administrationof these bifunctional ligands could be due to the differentialeffects of NOP activation depending on the site of action suchas spinal versus supraspinal. In rodents, spinal NOP pre-sumably mediates the analgesic effects, while the supraspinalNOP has antianalgesic effects (Meunier et al., 1995; Reinscheidet al., 1995). A possible explanation for the NOP antagonist-mediated enhancement in the antinociceptive effects ofsystemic BU08028 and SR16435 could be the inhibition ofMOP-mediated antinociception by supraspinal NOP receptors(Pan et al., 2000). However, the neurobiological mechanismsby which the supraspinal NOP receptors modulate antinoci-ception induced by spinal or systemic NOP or MOP agonistsare not fully understood.Buprenorphine is a speculated bifunctional ligand with

relatively high binding affinity at MOP and low bindingaffinity at NOP with partial agonist activity at MOP and NOPreceptors (Cowan et al., 1977; Huang et al., 2001; Clark et al.,2006). In humans, buprenorphine is a highly effective, long-acting opioid analgesic when administered via systemic,intrathecal, or epidural routes (Celleno and Capogna, 1989;Miwa et al., 1996; Pergolizzi et al., 2010), and it has a systemicpotency of 20 to 70 times that of morphine (Kress, 2009;Pergolizzi et al., 2010; Kawamoto et al., 2011). Our studieshave shown that intrathecal buprenorphine effectively pro-duces antihypersensitive effects against neuropathic andinflammatory pain. The antiallodynic effects of buprenorphinearemainlymediated byMOP receptors as spinally administered

Fig. 7. Effects of NOP and MOP antagonists on antiallodynic effects of intrathecal BU08028 (1 mg), SR16435 (3 mg) and buprenorphine (3 mg) inneuropathic pain at 0.5 hours. Antagonists J-113397 (3 mg), naltrexone (3 mg), or combination of naltrexone and J-113397 (3 mg) were intrathecallyadministered as a 10-minute pretreatment. Top panels: effects of antagonists on increased paw withdrawal thresholds (g) induced by BU08028 (A),SR16435 (C), and buprenorphine (E). Bottom panels: effects of antagonists on percentage of maximum possible antiallodynic effects of BU08028 (B),SR16435 (D), and buprenorphine (F). *Statistically significant difference from the vehicle control (P , 0.05).


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NOP antagonist did not alter buprenorphine’s antiallodyniceffects (Fig. 7). MOP-mediated antiallodynia may be expecteddue to its high binding affinity and intrinsic activity at MOPreceptors as well as MOP-mediated antinociception in rodents(Schmauss and Yaksh, 1984; Bernatzky and Jurna, 1986;Tejwani and Rattan, 2002) and nonhuman primates (Cremeanset al., 2012).However, it was recently documented in rodents that

antinociception produced by systemic buprenorphine is poten-tiated in the presence of systemically administered NOPantagonists and in NOP-receptor knockout mice (Lutfy et al.,2003; Ding and Raffa, 2009; Khroyan et al., 2009). Given thatbuprenorphine has an extremely low binding affinity at NOPversus MOP (Ki 5 285 versus 0.08 nM) and is much lesspotent in activating NOP receptors in vitro (EC50 5 35 versus0.08 nM) (Huang et al., 2001), it seems unlikely thatintrathecal buprenorphine activates the spinal NOP receptorsat antiallodynic doses. In particular, with well-justified dosesof MOP and NOP antagonists (Fig. 6), our data indicate that

buprenorphine’s susceptibility to NOP versus MOP antago-nism is different from the bifunctional NOP/MOP ligandsBU08028 and SR16435 (Fig. 7).In the clinical setting, development of tolerance to analge-

sia during long-term treatment with opioids is often reportedand poses a major challenge in pain management. The finalpart of the study compared development of tolerance to theantiallodynic effects of SR16435 and buprenorphine afterrepeated intrathecal administration (Fig. 8). Our hypothesiswas that a compound that simultaneously activates NOP andMOP receptors could have reduced or slower tolerance devel-opment as fewer receptors will be used to obtain antiallody-nia, thus keeping more receptors available for the subsequenttreatment. The antiallodynic effects of intrathecal buprenorphinedeclined relatively faster, with up to 35% reduction on day 5compared with day 1 of the treatment. A smaller change(12%) in the antiallodynic effects of intrathecal SR16435 wasobserved on day 5 as compared with day 1 of the treatment.These differences between SR16435 and buprenorphine could

Fig. 8. Development of tolerance to the antiallodynic effectsof repeated intrathecal administration of SR16435 (3 mg) orbuprenorphine (3 mg) in mice with neuropathic pain. (A)Changes in SR16435 and buprenorphine-induced increase inpawwithdrawal thresholds (g). (B) Changes in SR16435- andbuprenorphine-induced percentage of maximum possibleantiallodynic effects. Mice were tested each day at 0.5 hoursafter the drug administration.j is SR16435;d is buprenorphine.#Statistically significant difference from the antiallodyniceffects of SR16435 on day 1 of the treatment. *Statisticallydifference from the antiallodynic effects of buprenorphineon day 1 (P , 0.05).


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therefore be attributed to the additional NOP agonist activityof SR16435. Whether a bifunctional ligand with full agonistactivity at NOP andMOPwill have different rate and degree oftolerance development than a partial NOP/MOP agonist fortheir antihypersensitive effects is currently unknown. Never-theless, these findings indicate that bifunctional NOP/MOPagonists may provide effective spinal analgesia with a slowerdevelopment of tolerance.This study is the first to directly provide the pharmacolog-

ical evidence that intrathecally administered bifunctionalNOP/MOP ligands are not only as effective as but also morepotent than the selective full NOP or MOP agonists for theirantihypersensitive effects against neuropathic and inflamma-tory pain in mice. Studies in rodents and nonhuman primateshave previously shown that activation of NOP potentiatesMOP-mediated antinociception but not the MOP-associatedside effects such as motor dysfunction, respiratory depression,and pruritus (Tian et al., 1997; Ko and Naughton, 2009; Linand Ko, 2013; Sukhtankar and Ko, 2013). Analgesic drugstargeting both NOP and MOP receptors may therefore havethe desired improved efficacy and wider therapeutic window.Importantly, our data also indicate that such compounds mayhave a slower tolerance development to their analgesic effects.These findings further highlight the importance of investi-gating bifunctional NOP/MOP ligands as a novel therapeuticstrategy (Spagnolo et al., 2008; Toll et al., 2009; Cremeanset al., 2012; Molinari et al., 2013).Discovery of the NOP receptor crystal structure has

revealed the atomic details of ligand-receptor recognition(Thompson et al., 2012), which will facilitate the synthesis ofnovel compounds with optimum NOP/MOP-binding proper-ties to achieve effective analgesia with reduced side effectsand slower tolerance development. Together, our study demon-strates that at the spinal level in rodentsNOP receptorsmediatesimilar antihypersensitive effects as seen in nonhuman pri-mates and thus provides a platform to further validate theantinociceptive properties of bifunctional NOP/MOP ligandsin primatemodels. These preclinical studies therefore establisha translational bridge to the therapeutic profile of bifunctionalNOP/MOP ligands as spinal analgesics in humans.

Acknowledgments

The authors thank Matthew Zaks, Colette Cremeans, and ErinGruley for technical assistance with data collection.

Authorship Contributions

Participated in research design: Sukhtankar, Ko.Conducted experiments: Sukhtankar.Contributed new reagents: Zaveri, Husbands.Performed data analysis: Sukhtankar, Ko.Wrote or contributed to the writing of the manuscript: Sukhtankar,

Zaveri, Husbands, Ko.

References

Bennett GJ and Xie YK (1988) A peripheral mononeuropathy in rat that producesdisorders of pain sensation like those seen in man. Pain 33:87–107.

Bernatzky G and Jurna I (1986) Intrathecal injection of codeine, buprenorphine,tilidine, tramadol and nefopam depresses the tail-flick response in rats. Eur JPharmacol 120:75–80.

Briscini L, Corradini L, Ongini E, and Bertorelli R (2002) Up-regulation of ORL-1receptors in spinal tissue of allodynic rats after sciatic nerve injury. Eur J Phar-macol 447:59–65.

Calo G and Guerrini R (2013) Medicinal chemistry, pharmacology, and biologicalactions of peptide ligands selective for the NOP receptor, in Research and De-velopment of Opioid-Related Analgesics (Ko MC and Husbands SM, eds) pp275–325, ACS Books, Washington, DC.

Celleno D and Capogna G (1989) Spinal buprenorphine for postoperative analgesiaafter caesarean section. Acta Anaesthesiol Scand 33:236–238.

Chaplan SR, Bach FW, Pogrel JW, Chung JM, and Yaksh TL (1994) Quantitativeassessment of tactile allodynia in the rat paw. J Neurosci Methods 53:55–63.

Choi SS, Lee HK, Shim EJ, Kwon MS, Seo YJ, Lee JY, and Suh HW (2004) Alter-ations of c-Fos mRNA expression in hypothalamic-pituitary-adrenal axis andvarious brain regions induced by intrathecal single and repeated substance Padministrations in mice. Arch Pharm Res 27:863–866.

Clark MJ, Furman CA, Gilson TD, and Traynor JR (2006) Comparison of the relativeefficacy and potency of mu-opioid agonists to activate Ga(i/o) proteins containinga pertussis toxin-insensitive mutation. J Pharmacol Exp Ther 317:858–864.

Courteix C, Coudoré-Civiale MA, Privat AM, Pélissier T, Eschalier A, and Fialip J(2004) Evidence for an exclusive antinociceptive effect of nociceptin/orphanin FQ,an endogenous ligand for the ORL1 receptor, in two animal models of neuropathicpain. Pain 110:236–245.

Cowan A, Lewis JW, and Macfarlane IR (1977) Agonist and antagonist properties ofbuprenorphine, a new antinociceptive agent. Br J Pharmacol 60:537–545.

Cremeans CM, Gruley E, Kyle DJ, and Ko MC (2012) Roles of m-opioid receptors andnociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiologicalresponses in primates. J Pharmacol Exp Ther 343:72–81.

Davis AM and Inturrisi CE (1999) D-Methadone blocks morphine tolerance and N-methyl-D-aspartate-induced hyperalgesia. J Pharmacol Exp Ther 289:1048–1053.

Ding Z and Raffa RB (2009) Identification of an additional supraspinal component tothe analgesic mechanism of action of buprenorphine. Br J Pharmacol 157:831–843.

Dixon WJ (1980) Efficient analysis of experimental observations. Annu Rev Phar-macol Toxicol 20:441–462.

Fairbanks CA (2003) Spinal delivery of analgesics in experimental models of painand analgesia. Adv Drug Deliv Rev 55:1007–1041.

Hao JX, Xu IS, Wiesenfeld-Hallin Z, and Xu XJ (1998) Anti-hyperalgesic and anti-allodynic effects of intrathecal nociceptin/orphanin FQ in rats after spinal cordinjury, peripheral nerve injury and inflammation. Pain 76:385–393.

Hopkins E, Rossi G, and Kest B (2004) Sex differences in systemic morphine anal-gesic tolerance following intrathecal morphine injections. Brain Res 1014:244–246.

Hu E, Calò G, Guerrini R, and Ko MC (2010) Long-lasting antinociceptive spinaleffects in primates of the novel nociceptin/orphanin FQ receptor agonist UFP-112.Pain 148:107–113.

Huang P, Kehner GB, Cowan A, and Liu-Chen LY (2001) Comparison of pharma-cological activities of buprenorphine and norbuprenorphine: norbuprenorphine isa potent opioid agonist. J Pharmacol Exp Ther 297:688–695.

Jia Y, Linden DR, Serie JR, and Seybold VS (1998) Nociceptin/orphanin FQ bindingincreases in superficial laminae of the rat spinal cord during persistent peripheralinflammation. Neurosci Lett 250:21–24.

Kawamoto S, Tatsumi K, Kataoka T, Kamikawa T, Yanagida T, and Mandai R (2011)Comparison of intrathecal morphine and buprenorphine for postoperative anal-gesia in cesarean delivery. [article in Japanese]. Masui 60:892–896.

Khroyan TV, Polgar WE, Cami-Kobeci G, Husbands SM, Zaveri NT, and Toll L(2011a) The first universal opioid ligand, (2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol (BU08028): characterization of the in vitro profile and in vivobehavioral effects in mouse models of acute pain and cocaine-induced reward. JPharmacol Exp Ther 336:952–961.

Khroyan TV, Polgar WE, Jiang F, Zaveri NT, and Toll L (2009) Nociceptin/orphaninFQ receptor activation attenuates antinociception induced by mixed nociceptin/orphanin FQ/mu-opioid receptor agonists. J Pharmacol Exp Ther 331:946–953.

Khroyan TV, Polgar WE, Orduna J, Montenegro J, Jiang F, Zaveri NT, and Toll L(2011b) Differential effects of nociceptin/orphanin FQ (NOP) receptor agonists inacute versus chronic pain: studies with bifunctional NOP/m receptor agonists in thesciatic nerve ligation chronic pain model in mice. J Pharmacol Exp Ther 339:687–693.

Khroyan TV, Zaveri NT, Polgar WE, Orduna J, Olsen C, Jiang F, and Toll L (2007)SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a novelmixed nociceptin/orphanin FQ/mu-opioid receptor partial agonist: analgesic andrewarding properties in mice. J Pharmacol Exp Ther 320:934–943.

Ko MC and Naughton NN (2009) Antinociceptive effects of nociceptin/orphanin FQadministered intrathecally in monkeys. J Pain 10:509–516.

Ko MC, Woods JH, Fantegrossi WE, Galuska CM, Wichmann J, and Prinssen EP(2009) Behavioral effects of a synthetic agonist selective for nociceptin/orphanin FQpeptide receptors in monkeys. Neuropsychopharmacology 34:2088–2096.

Kress HG (2009) Clinical update on the pharmacology, efficacy and safety of trans-dermal buprenorphine. Eur J Pain 13:219–230.

Lambert DG (2008) The nociceptin/orphanin FQ receptor: a target with broad ther-apeutic potential. Nat Rev Drug Discov 7:694–710.

Largent-Milnes TM and Vanderah TW (2010) Recently patented and promising ORL-1 ligands: where have we been and where are we going? Expert Opin Ther Pat 20:291–305.

Lewis JW (1985) Buprenorphine. Drug Alcohol Depend 14:363–372.Lin AP and Ko MC (2013) The therapeutic potential of nociceptin/orphanin FQ re-ceptor agonists as analgesics without abuse liability. ACS Chem Neurosci 4:214–224 DOI: .

Lutfy K, Eitan S, Bryant CD, Yang YC, Saliminejad N, Walwyn W, Kieffer BL,Takeshima H, Carroll FI, and Maidment NT, et al. (2003) Buprenorphine-inducedantinociception is mediated by mu-opioid receptors and compromised by concom-itant activation of opioid receptor-like receptors. J Neurosci 23:10331–10337.

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL,Guillemot JC, Ferrara P, and Monsarrat B, et al. (1995) Isolation and structure ofthe endogenous agonist of opioid receptor-like ORL1 receptor.Nature 377:532–535.

Miwa Y, Yonemura E, and Fukushima K (1996) Epidural administered buprenor-phine in the perioperative period. Can J Anaesth 43:907–913.

Molinari S, Camarda V, Rizzi A, Marzola G, Salvadori S, Marzola E, Molinari P,McDonald J, Ko MC, and Lambert DG, et al. (2013) [Dmt1]N/OFQ(1-13)-NH2:


at ASPE

T Journals on O


Dow

nloaded from


a potent nociceptin/orphanin FQ and opioid receptor universal agonist. Br JPharmacol 168:151–162.

Obara I, Przewlocki R, and Przewlocka B (2005) Spinal and local peripheral anti-allodynic activity of Ro64-6198 in neuropathic pain in the rat. Pain 116:17–25.

Pan Z, Hirakawa N, and Fields HL (2000) A cellular mechanism for the bidirectionalpain-modulating actions of orphanin FQ/nociceptin. Neuron 26:515–522.

Pergolizzi J, Aloisi AM, Dahan A, Filitz J, Langford R, Likar R, Mercadante S,Morlion B, Raffa RB, and Sabatowski R, et al. (2010) Current knowledge ofbuprenorphine and its unique pharmacological profile. Pain Pract 10:428–450.

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR,Grandy DK, Langen H, Monsma FJ, Jr, and Civelli O (1995) Orphanin FQ:a neuropeptide that activates an opioidlike G protein-coupled receptor. Science270:792–794.

Reiss D, Wichmann J, Tekeshima H, Kieffer BL, and Ouagazzal AM (2008) Effects ofnociceptin/orphanin FQ receptor (NOP) agonist, Ro64-6198, on reactivity to acutepain in mice: comparison to morphine. Eur J Pharmacol 579:141–148.

Schmauss C and Yaksh TL (1984) In vivo studies on spinal opiate receptor systemsmediating antinociception. II. Pharmacological profiles suggesting a differentialassociation of mu, delta and kappa receptors with visceral chemical and cutaneousthermal stimuli in the rat. J Pharmacol Exp Ther 228:1–12.

Sounvoravong S, Takahashi M, Nakashima MN, and Nakashima K (2004) Disabilityof development of tolerance to morphine and U-50,488H, a selective kappa-opioidreceptor agonist, in neuropathic pain model mice. J Pharmacol Sci 94:305–312.

Spagnolo B, Calo G, Polgar WE, Jiang F, Olsen CM, Berzetei-Gurske I, Khroyan TV,Husbands SM, Lewis JW, and Toll L, et al. (2008) Activities of mixed NOP and mu-opioid receptor ligands. Br J Pharmacol 153:609–619.

Sukhtankar DS and Ko MC (2013) Pharmacological investigation of NOP-relatedligands as analgesics without abuse liability, in Research and Development of

Opioid-Related Analgesics (Ko MC and Husbands SM, eds) pp 393–416, ACSBooks, Washington, DC.

Tejwani GA and Rattan AK (2002) The role of spinal opioid receptors in anti-nociceptive effects produced by intrathecal administration of hydromorphone andbuprenorphine in the rat. Anesth Analg 94:1542–1546 table of contents.

Thompson AA, Liu W, Chun E, Katritch V, Wu H, Vardy E, Huang XP, Trapella C,Guerrini R, and Calo G, et al. (2012) Structure of the nociceptin/orphanin FQreceptor in complex with a peptide mimetic. Nature 485:395–399.

Tian JH, Xu W, Fang Y, Mogil JS, Grisel JE, Grandy DK, and Han JS (1997) Bi-directional modulatory effect of orphanin FQ on morphine-induced analgesia: an-tagonism in brain and potentiation in spinal cord of the rat. Br J Pharmacol 120:676–680.

Toll L, Berzetei-Gurske IP, Polgar WE, Brandt SR, Adapa ID, Rodriguez L, SchwartzRW, Haggart D, O’Brien A, and White A, et al. (1998) Standard binding andfunctional assays related to medications development division testing for potentialcocaine and opiate narcotic treatment medications. NIDA Res Monogr 178:440–466.

Toll L, Khroyan TV, Polgar WE, Jiang F, Olsen C, and Zaveri NT (2009) Comparisonof the antinociceptive and antirewarding profiles of novel bifunctional nociceptinreceptor/mu-opioid receptor ligands: implications for therapeutic applications.J Pharmacol Exp Ther 331:954–964.

Address correspondence to: Dr. Mei-Chuan Ko, Department of Physiologyand Pharmacology, Wake Forest University School of Medicine, MedicalCenter Boulevard, NRC 205, Winston-Salem, NC 27157. E-mail: [email protected]


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